Affinity-based partition assay for detection of target molecules

• US 9,850,515 B2
• Filed: 02/05/2014
• Issued: 12/26/2017
• Est. Priority Date: 02/08/2013
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: McCoy, Adam M., Tzonev, Svilen, Hsiung, Frank
• Primary Examiner(s): Cheu, Jacob
• Application Number:
  US14/173,562
• Publication Number:
  US 20140228239A1
• Time in Patent Office: 1,420 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The present invention provides methods of detecting a target molecule in a sample comprising incubating the sample with two or more detectably labeled probes, partitioning the sample into multiple partitions, and detecting the presence of the two or more probes in the same partition.

Claims

1. A method of detecting a target molecule in a sample, the method comprising:

incubating said sample in a mixture with at least a first probe linked to a first label and a second probe linked to a second label, wherein the first and second probes specifically bind the same target molecule, if present;

partitioning the mixture into two or more partitions, wherein on average the partitions have fewer than two probes; and

detecting the presence of the first label and the second label in at least one same partition, thereby detecting the target molecule in the sample.

2. The method of claim 1, wherein the partitioning comprises generating a sufficient number of partitions such that co-localization of the probes due to binding of a target molecule can be distinguished from random co-localization.

3. The method of claim 1, wherein the first and second labels comprise nucleic acids, fluorescent moieties, affinity tags, or click chemistry moieties, and wherein the first label is different from the second label.

4. The method of claim 1, wherein the first label generates a first signal and the second label generates a second signal and the first signal and the second signal are distinguishable.

5. The method of claim 4, wherein the first label is a first enzyme and the second label is a second enzyme, and the detecting comprises detecting products generated by the first and second enzymes.

6. The method of claim 1, wherein the first label and the second label combine to produce a signal that is not generated in the absence of the first label, the second label, or both.

7. The method of claim 6, wherein the first label is a first enzyme and the second label is a second enzyme and the activities of the first and second enzymes combine to generate a detectable signal indicative of the presence of the first and second labels.

8. The method of claim 1, wherein the signal from the first label and the signal from the second label are amplified following the partitioning.

9. The method of claim 1, further comprising determining the number of partitions comprising the first label and the second label.

10. The method of claim 1, wherein the partitions are droplets or microchannels.

11. The method of claim 1, wherein the target molecule is a protein or a nucleic acid.

12. The method of claim 1, wherein the first probe and/or the second probe comprises (i) a binding agent independently selected from the group consisting of antibodies, aptamers, and non-antibody protein scaffolds, or (ii) a target-specific binding agent independently selected from the group consisting of a nucleic acid and a zinc finger protein.

13. A method of detecting a target molecule in a sample, wherein the target molecule is linked to, or is capable of generating, a detectable molecule, the method comprising:

incubating the sample in a mixture with a first probe linked to a first label, wherein the first probe specifically binds the target molecule, if present;

partitioning the mixture into two or more partitions, wherein on average the partitions have fewer than one probe; and

detecting the presence of the first label and the detectable molecule in at least one same partition, thereby detecting the target molecule in the sample.

14. The method of claim 13, wherein the target molecule is an enzyme capable of converting a substrate in the mixture into the detectable molecule or into an intermediate molecule that is further converted into the detectable molecule by the first label.